ClpL

ABSTRACT

The invention provides clpL polypeptides and DNA (RNA) encoding clpL polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing clpL polypeptides to screen for antibacterial compounds.

This application claims the benefit of U.S. Provisional Application No.60/011,888, filed Feb. 20, 1996 which is a con of internationalapplication PCT/US97/02318, filed Feb. 19, 1997.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, in these and inother regards, the invention relates to novel polynucleotides andpolypeptides of the ATP dependent proteases family, hereinafter referredto as “clpL”.

BACKGROUND OF THE INVENTION

It is particularly preferred to employ Staphylococcal genes and geneproducts as targets for the development of antibiotics. TheStaphylococci make up a medically important genera of microbes. They areknown to produce two types of disease, invasive and toxigenic. Invasiveinfections are characterized generally by abscess formation effectingboth skin surfaces and deep tissues. Staphylococcus aureus is the secondleading cause of bacteremia in cancer patients. Osteomyelitis, septicarthritis, septic thrombophlebitis and acute bacterial endocarditis arealso relatively common. These are at least clinical conditions resultingfrom the toxigenic properties of Staphylococci. The manifestation ofthese diseases result from the actions of exotoxins as opposed to tissueinvasion and bacteremia. These conditions include: Staphylococcal foodpoisoning, scalded skin syndrome and toxic shock syndrome.

The frequency of Staphylococcus aureus infections has risen dramaticallyin the past 20 years. This has been attributed to the emergence ofmultiply antibiotic resistant strains and an increasing population ofpeople with weakened immune systems. It is no longer uncommon to isolateStaphylococcus aureus strains which are resistant to some or all of thestandard antibiotics. This has created a demand for both newanti-microbial agents and diagnostic tests for this organism.

Recently several novel approaches have been described which purport tofollow global gene expression during infection (Chuang, S. et al.,(1993); Mahan, M. J. et al., Science 259:686-688 (1993); Hensel, M. etal., Science 269:400-403 (1995). These new techniques have so far beendemonstrated with gram negative pathogen infections, but not withinfections with gram positives presumably because the much slowerdevelopment of global transposon mutagenesis and suitable vectors neededfor these strategies in these organisms, and in the case of that processdescribed by Chuang, S. et al., J. Bacteriol. 175:2026-2036 (1993), thedifficulty of isolating suitable quantities of bacterial RNA free ofmammalian RNA derived from the infected tissue to furnish bacterial RNAlabelled to sufficiently high specific activity.

The present invention employs a novel technology to determine geneexpression in the pathogen at different stages of infection of themammalian host. A novel aspect of this invention is the use of asuitably labelled oligonucleotide probe which anneals specifically tothe bacterial ribosomal RNA in Northern blots of bacterial RNApreparations from infected tissue. Using the more abundant ribosomal RNAas a hybridization target greatly facilitates the optimization of aprotocol to purify bacterial RNA of a suitable size and quantity forRT-PCR from infected tissue.

A suitable oligonucleotide useful for applying this method to genesexpressed in Staphylococcus aureus is 5′-GCTCCTAAAAGGTTACTCCACCGGC-3′[SEQ ID NO: 5].

Use of the technology of the present invention enables identification ofbacterial genes transcribed during infection, inhibitors of which wouldhave utility in anti-bacterial therapy. Specific inhibitors of such genetranscription or of the subsequent translation of the resultant mRNA orof the function of the corresponding expressed proteins would haveutility in anti-bacterial therapy. This invention providesStaphylococcus aureus WCUH29 polynucleotides which are transcribed ininfected tissue.

Clearly, there is a need for factors, such as the novel compounds of theinvention, that have a present benefit useful to screen compounds forantibiotic activity. Such factors are also useful to determine theirrole in pathogenesis of infection, dysfunction and disease. There isalso a need for identification and characterization of such factors andtheir antagonists and agonists which can play a role in preventing,ameliorating or correcting infections, dysfunctions or diseases.

The polypeptides of the invention have amino acid sequence homology to aknown Lactococcus lactis ClpL protein (Genbank Accession Number:X62333).

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have beenidentified as novel clpL polypeptides by homology between the amino acidsequence set out in Table 1 [SEQ ID NO: 2] and a known amino acidsequence or sequences of other proteins such as Lactococcus lactis ClpLprotein.

It is a further object of the invention to provide polynucleotides thatencode clpL polypeptides, particularly polynucleotides that encode thepolypeptide herein designated clpL.

In a particularly preferred embodiment of the invention, thepolynucleotide comprises a region encoding clpL polypeptides comprisingthe sequence set out in Table 1 [SEQ ID NO:1] which includes a fulllength gene, or a variant thereof.

In another particularly preferred embodiment of the invention, there isa novel clpL protein from Staphylococcus aureus comprising the aminoacid sequence of Table 1 [SEQ ID NO:2], or a variant thereof.

In accordance with another aspect of the invention, there is provided anisolated nucleic acid molecule encoding a mature polypeptide expressibleby the Staphylococcus aureus WCUH 29 strain contained in the depositedstrain.

As a further aspect of the invention, there are provided isolatednucleic acid molecules encoding clpL, particularly Staphylococcus aureusclpL, including mRNAs, cDNAs, genomic DNAs. Further embodiments of theinvention include biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

In accordance with another aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of clpL and polypeptides encoded thereby.

As another aspect of the invention, there are provided novelpolypeptides of Staphylococcus aureus referred to herein as clpL as wellas biologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

Among the particularly preferred embodiments of the invention arevariants of clpL polypeptide encoded by naturally occurring alleles ofthe clpL gene.

In a preferred embodiment of the invention, there are provided methodsfor producing the aforementioned clpL polypeptides.

In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

In accordance with certain preferred embodiments of the invention, thereare provided products, compositions and methods for assessing clpLexpression, treating disease, for example, disease, such as, infectionsof the upper respiratory tract (e.g., otitis media, bacterialtracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g.empyema, lung abscess), cardiac (e.g., infective endocarditis),gastrointestinal (e.g., secretory diarrhoea, splenic absces,retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g.,blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal andorbital cellulitis, darcryocystitis), kidney and urinary tract (e.g.,epididymitis, intrarenal and perinephric absces, toxic shock syndrome),skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis,wound infection, bacterial myositis) bone and joint (e.g., septicarthritis, osteomyelitis), assaying genetic variation, and administeringa clpL polypeptide or polynucleotide to an organism to raise animmunological response against a bacteria, especially a Staphylococcusaureus bacteria.

In accordance with certain preferred embodiments of this and otheraspects of the invention, there are provided polynucleotides thathybridize to clpL polynucleotide sequences, particularly under stringentconditions.

In certain preferred embodiments of the invention, there are providedantibodies against clpL polypeptides.

In other embodiments of the invention, there are provided methods foridentifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor polynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypeptideor polynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

In accordance with yet another aspect of the invention, there areprovided clpL agonists and antagonists, preferably bacteriostatic orbacteriocidal agonists and antagonists.

In a further aspect of the invention, there are provided compositionscomprising a clpL polynucleotide or a clpL polypeptide foradministration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

GLOSSARY

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

“Host” is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, NewYork, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). Preferred methods to determine identity are designed togive the largest match between the sequences tested. Methods todetermine identity and similarity are codified in publicly availablecomputer programs. Preferred computer program methods to determineidentity and similarity between two sequences include, but are notlimited to, the GCG program package (Devereux, J., et al., Nucleic AcidsResearch 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F.et al., J. Molec. Biol. 215: 403-410 (1990). The BLAST X program ispublicly available from NCBI and other sources (BLAST Manual, Altschul,S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J.Mol. Biol. 215: 403-410 (1990). As an illustration, by a polynucleotidehaving a nucleotide sequence having at least, for example, 95%“identity” to a reference necleotide sequence of SEQ ID NO: 1 it isintended that the nucleotide sequence of the polynucleotide is identicalto the reference sequence except that the polynucleotide sequence mayinclude up to five point mutations per each 100 nucleotides of thereference nucleotide sequence of SEQ ID NO: 1. In other words, to obtaina polynucleotide having a nucleotide sequence at least 95% identical toa reference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence.These mutations of the reference sequence may occur at the 5 or 3terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence. Analogously, by a polypeptidehaving an amino acid sequence having at least, for example, 95% identityto a reference amino acid sequence of SEQ ID NO:2 is intended that theamino acid sequence of the polypeptide is identical to the referencesequence except that the polypeptide sequence may include up to fiveamino acid alterations per each 100 amino acids of the reference aminoacid of SEQ ID NO: 2. In other words, to obtain a polypeptide having anamino acid sequence at least 95% identical to a reference amino acidsequence, up to 5% of the amino acid residues in the reference sequencemay be deleted or substituted with another amino acid, or a number ofamino acids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated, ”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein.

“Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- or double-strandedregions or single-, double- and triple-stranded regions, single- anddouble-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term“polynucleotides(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

“Polypeptide(s)” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. “Polypeptide(s)” refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene coded amino acids. “Polypeptide(s)” include thosemodified by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino of carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched or branched circular polypeptides may resultfrom post-translational natural processes and may be made by entirelysynthetic methods, as well.

“Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

DESCRIPTION OF THE INVENTION

The invention relates to novel clpL polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of a novel clpL of Staphylococcusaureus, which is related by amino acid sequence homology to Lactococcuslactis ClpL polypeptide (Lactococcus lactis clpL Genbank AccessionNumber: X62333). The invention relates especially to clpL having thenucleotide and amino acid sequences set out in Table 1 [SEQ ID NO: 1]and Table 1 [SEQ ID NO: 2] respectively, and to the clpL nucleotidesequences of the DNA in and deposited strain and amino acid sequencesencoded thereby.

TABLE 1 clpL Polynucleotide and Polypeptide Sequences (A) Sequences fromStaphylococcus aureus clpL polynucleotide sequence [SEQ ID NO: 1]. 5′-1ATGAATAACG GTTTTTTCAA TAGCGACTTT GATTCAATTT TTCGAAGAAT 51 GATGCAAGATATGCAAGGTT CAAATCAAGT CGGTAACAAA AAGTACTATA 101 TTAATGGTAA AGAAGTTTCACCTGAAGAAC TAGCGCAACT CACACAACAA 151 GGTAGCAATC AATCTGCTGA ACAAAGTGCGCAAGCTTTTC AACAAGCAGC 201 ACAAAGACAA CAAGGGCAAC AAGGTGGCAA CGGCAATTATTTAGAACAAA 251 TTGGTCGTAA CCTTACGCAA GAAGCACGTG ACGGTTTATT AGATCCAGTC301 ATTGGTCGTG ATAAAGAAAT TCAAGAAACT GCTGAAGTCT TAAGTAGACG 351AACTAAAAAC AATCCTATAT TAGTTGGAGA AGCTGGTGTT GGTAAAACTG 401 CGATTGTTGAAGGTTTAGCA CAGGCAATCG TTGAAGGAAA TGTACCAGCA 451 GCAATCAAAG ACAAAGAAATTATTTCTGTA GATATTTCAT CATTAGAAGC 501 TGGAACGCAA TATCGTGGTG CTTTTGAAGAAAATATTCAA AAATTAATCG 551 AAGGTGTTAA ATCTTCACAA AATGCCGTAC TATTCTTTGATGAAATCCAT 601 CAAATTATCG GTTCAGGTGC CACAGGAAGT GATTCAGGTA GCAAAGGGTT651 ATCTGATATT TTGAAACCTG CATTAAGTCG TGGTGAGATT TCTATTATTG 701GTGCAACAAC ACAAGATGAA TATCGAAACA ATATTCTTAA AGATGCTGCA 751 TTAACGCGCAGATTTAATGA AGTGCTTGTT AATGAACCAA GCGCTAAAGA 801 TACTGTTGAA ATTTTAAAAGGTATTCGCGA AAAATTCGAA GAACACCATC 851 AAGTAAAATT ACCAGATGAC GTATTAAAAGCATGTGTTGA CTTATCAATT 901 CAATATATTC CACAACGATT ATTACCAGAT AAAGCAATCGATGTGTTAGA 951 TATTACAGCA GCACATTTAT CTGCGCAAAG TCCAGCTGTC GATAAAGTTG1001 AAACTGAAAA ACGAATTTCT GAATTAGAAA ATGATAAACG TAAAGCAGTA 1051AGTGCTGAAG AATATAAAAA AGCTGACGAC ATTCAAAATG AAATCAAATC 1101 ATTACAAGATAAATTAGAAA ACAGTAATGG TGAACATACT GCTGTTGCTA 1151 CAGTTCATGA TATTTCAGATACTATTCAAC GATTAACTGG CATTCCAGTT 1201 TCTCAAATGG ATGATAACGA TATTGAACGTTTAAAAAATA TTTCTAATCG 1251 TTTAAGAAGT AAAATCATAG GTCAAGATCA AGCTGTAGAAATGGTTTCAC 1301 GTGCAATTCG CCGTAATCGC GCTGGGTTTG ATGACGGCAA CCGTCCAATT1351 GGTAGTTTCT TATTTGTCGG CCCTACTGGT GTTGGTAAAA CAGAGCTTGC 1401TAAACAATTA GCAATTGATC TATTTGGTAA TAAAGATGCA CTTATTCGAC 1451 TTGATATGAGTGAATATAGT GACACAACAG CTGTTTCAAA AATGATTGGT 1501 ACAACTGCTG GTTATGTCGGTTATGATGAC AATTCAAATA CGTTAACTGA 1551 AAAAGTACGC CGTAATCCAT ACTCAGTCATTCTATTTGAT GAAATCGAAA 1601 AAGCAAATCC ACAAATTTTA ACATTGTTAT TACAAGTAATGGATGATGGT 1651 AATTTGACTG ATGGTCAAGG TAATGTCATC AACTTTAAAA ATACAATTAT1701 TATTTGTACA TCAAATGCTG GCTTTGGCAA TGGCAATGAC GCTGAAGAAA 1751AAGATATTAT GCACGAAATG AAAAAATTCT TCCGCCCTGA ATTCCTTAAC 1801 CGCTTCAACGGCATCGTTGA ATTCTTACAT TTAGATAAAG ATGCATTGCA 1851 AGATATCGTC AACTTATTATTAGACGATGT ACAAGTTACA TTAGATAAAA 1901 AAGGTATTAC GATGGACGTT TCTCAAGATGCGAAAGATTG GTTAATTGAA 1951 GAAGGCTATG ATGAAGAATT AGGTGCACGT CCATTAAGACGTATTGTTGA 2001 ACAGCAAGTA CGTGACAAAA TTACAGATTA TTATCTAGAT CATACAGACG2051 TTAAACATGT GGATATAGAT GTTGAGGATA ACGAATTAGT CGTAAAAGGT 2101AAATAA-3′ (B) clpL polypeptide sequence deduced from the polynucleotidesequence in this table [SEQ ID NO:2]. NH₂-1 MNGFFNSDF DSIFRRMMQDMQGSNQVGNK KYYINGKEVS PEELAQLTQQ 51 GSNQSAEQSA QAFQQAAQRQ QGQQGGNGNYLEQIGRNLTQ EARDGLLDPV 101 IGRDKEIQET AEVLSRRTKN NPILVGEAGV GKTAIVEGLAQAIVEGNVPA 151 AIKDKEIISV DISSLEAGTQ YRGAFEENIQ KLIEGVKSSQ NAVLFFDEIH201 QIIGSGATGS DSGSKGLSDI LKPALSRGEI SIIGATTQDE YRNNILKDAA 251LTRRFNEVLV NEPSAKDTVE ILKGIREKFE EHHQVKLPDDVLKACVDLSI 301 QYIPQRLLFDKAIDVLDITA AHLSAQSPAV DKVETEKRIS ELENDKRKAV 351 SAEEYKKADD IQNEIKSLQDKLENSNGEHT AVATVHDISD TIQRLTGIPV 401 SQMDDNDIER LKNISNRLRS KIIGQDQAVEMVSRAIRRNR AGFDDGNRPI 451 GSFLFVGPTG VGKTELAKQL AIDLFGNKDA LIRLDMSEYSDTTAVSKMIG 501 TTAGYVGYDD NSNTLTEKVR RNPYSVILFD EIEKANPQIL TLLLQVMDDG551 NLTDGQGNVI NFKNTIIICT SNAGFGNGND AEEKDIMHEM KKFFRPEFLN 601RFNGIVEFLH LDKDALQDIV NLLLDDVQVT LDKKGITMDV SQDAKDWLIE 651 EGYDEELGARPLRRIVEQQV RDKITDYYLD HTDVKHVDID VEDNELVVKG 701 K-COOH (C)Polynucleotide sequence embodiments [SEQ ID NO: 1]. X-(R₁)_(n)-1ATGAATAACG GTTTTTTCAA TAGCGACTTT GATTCAATTT TTCGAAGAAT 51 GATGCAAGATATGCAAGGTT CAAATCAAGT CGGTAACAAA AAGTACTATA 101 TTAATGGTAA AGAAGTTTCACCTGAAGAAC TAGCGCAACT CACACAACAA 151 GGTAGCAATC AATCTGCTGA ACAAAGTGCGCAAGCTTTTC AACAAGCAGC 201 ACAAAGACAA CAAGGGCAAC AAGGTGGCAA CGGCAATTATTTAGAACAAA 251 TTGGTCGTAA CCTTACGCAA GAAGCACGTG ACGGTTTATT AGATCCAGTC301 ATTGGTCGTG ATAAAGAAAT TCAAGAAACT GCTGAAGTCT TAAGTAGACG 351AACTAAAAAC AATCCTATAT TAGTTGGAGA AGCTGGTGTT GGTAAAACTG 401 CGATTGTTGAAGGTTTAGCA CAGGCAATCG TTGAAGGAAA TGTACCAGCA 451 GCAATCAAAG ACAAAGAAATTATTTCTGTA GATATTTCAT CATTAGAAGC 501 TGGAACGCAA TATCGTGGTG CTTTTGAAGAAAATATTCAA AAATTAATCG 551 AAGGTGTTAA ATCTTCACAA AATGCCGTAC TATTCTTTGATGAAATCCAT 601 CAAATTATCG GTTCAGGTGC CACAGGAAGT GATTCAGGTA GCAAAGGGTT651 ATCTGATATT TTGAAACCTG CATTAAGTCG TGGTGAGATT TCTATTATTG 701GTGCAACAAC ACAAGATGAA TATCGAAACA ATATTCTTAA AGATGCTGCA 751 TTAACGCGCAGATTTAATGA AGTGCTTGTT AATGAACCAA GCGCTAAAGA 801 TACTGTTGAA ATTTTAAAAGGTATTCGCGA AAAATTCGAA GAACACCATC 851 AAGTAAAATT ACCAGATGAC GTATTAAAAGCATGTGTTGA CTTATCAATT 901 CAATATATTC CACAACGATT ATTACCAGAT AAAGCAATCGATGTGTTAGA 951 TATTACAGCA GCACATTTAT CTGCGCAAAG TCCAGCTGTC GATAAAGTTG1001 AAACTGAAAA ACGAATTTCT GAATTAGAAA ATGATAAACG TAAAGCAGTA 1051AGTGCTGAAG AATATAAAAA AGCTGACGAC ATTCAAAATG AAATCAAATC 1101 ATTACAAGATAAATTAGAAA ACAGTAATGG TGAACATACT GCTGTTGCTA 1151 CAGTTCATGA TATTTCAGATACTATTCAAC GATTAACTGG CATTCCAGTT 1201 TCTCAAATGG ATGATAACGA TATTGAACGTTTAAAAAATA TTTCTAATCG 1251 TTTAAGAAGT AAAATCATAG GTCAAGATCA AGCTGTAGAAATGGTTTCAC 13O1 GTGCAATTCG CCGTAATCGC GCTGGGTTTG ATGACGGCAA CCGTCCAATT1351 GGTAGTTTCT TATTTGTCGG CCCTACTGGT GTTGGTAAAA CAGAGCTTGC 1401TAAACAATTA GCAATTGATC TATTTGGTAA TAAAGATGCA CTTATTCGAC 1451 TTGATATGAGTGAATATAGT GACACAACAG CTGTTTCAAA AATGATTGGT 1501 ACAACTGCTG GTTATGTCGGTTATGATGAC AATTCAAATA CGTTAACTGA 1551 AAAAGTACGC CGTAATCCAT ACTCAGTCATTCTATTTGAT GAAATCGAAA 1601 AAGCAAATCC ACAAATTTTA ACATTGTTAT TACAAGTAATGGATGATGGT 1651 AATTTGACTG ATGGTCAAGG TAATGTCATC AACTTTAAAA ATACAATTAT1701 TATTTGTACA TCAAATGCTG GCTTTGGCAA TGGCAATGAC GCTGAAGAAA 1751AAGATATTAT GCACGAAATG AAAAAATTCT TCCGCCCTGA ATTCCTTAAC 1801 CGCTTCAACGGCATCGTTGA ATTCTTACAT TTAGATAAAG ATGCATTGCA 1851 AGATATCGTC AACTTATTATTAGACGATGT ACAAGTTACA TTAGATAAAA 1901 AAGGTATTAC GATGGACGTT TCTCAAGATGCGAAAGATTG GTTAATTGAA 1951 GAAGGCTATG ATGAAGAATT AGGTGCACGT CCATTAAGACGTATTGTTGA 2001 ACAGCAAGTA CGTGACAAAA TTACAGATTA TTATCTAGAT CATACAGACG2051 TTAAACATGT GGATATAGAT GTTGAGGATA ACGAATTAGT CGTAAAAGGT 2101AAATAA-(R₂)_(n)-Y (D) Polypeptide sequence embodiments [SEQ ID NO:2].X-(R₁)_(n)-1 MNNGFFNSDF DSIFRRMMQD MQGSNQVGNK KYYINGKEVS PEELAQLTQQ 51GSNQSAEQSA QAFQQAAQRQ QGQQGGNGNY LEQIGRNLTQ EARDGLLDPV 101 IGRDKEIQETAEVLSRRTKN NPILVGEAGV GKTAIVEGLA QAIVEGNVPA 151 AIKDKEIISV DISSLEAGTQYRGAFEENIQ KLIEGVKSSQ NAVLFFDEIH 201 QIIGSGATGS DSGSKGLSDI LKPALSRGEISIIGATTQDE YRNNILKDAA 251 LTRRFNEVLV NEPSAKDTVE ILKGIREKFE EHHQVKLPDDVLKACVDLSI 301 QYIPQRLLPD KAIDVLDITA AHLSAQSPAV DKVETEKRIS ELENDKRKAV351 SAEEYKKADD IQNEIKSLQD KLENSNGEHT AVATVHDISD TIQRLTGIPV 401SQMDDNDIER LKNISNRLRS KIIGQDQAVE MVSRAIRRNR AGFDDGNRPI 451 GSFLFVGPTGVGKTELAKQL AIDLFGNKDA LIRLDMSEYS DTTAVSKNIG 501 TTAGYVGYDD NSNTLTEKVRRNPYSVILFD EIEKANPQIL TLLLQVMDDG 551 NLTDGQGNVI NFKNTIIICT SNAGFGNGNDAEEKDIMHEM KKFFRPEFLN 601 RFNGIVEFLH LDKDALQDIV NLLLDDVQVT LDKKGITMDVSQDAKDWLIE 651 EGYDEELGAR PLRRIVEQQV RDKITDYYLD HTDVKHVDID VEDNELVVKG701 K-(R₂)_(n)-Y

Deposited materials

A deposit containing a Staphylococcus aureus WCUH 29 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein “NCIMB”), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on Sep. 11, 1995 and assigned NCIMB Deposit No. 40771, andreferred to as Staphylococcus aureus WCUH29 on deposit. TheStaphylococcus aureus strain deposit is referred to herein as “thedeposited strain” or as “the DNA of the deposited strain.”

The deposited strain contains the full length clpL gene. The sequence ofthe polynucleotides contained in the deposited strain, as well as theamino acid sequence of the polypeptide encoded thereby, are controllingin the event of any conflict with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

A license may be required to make, use or sell the deposited strain, andcompounds derived therefrom, and no such license is hereby granted.

Polypeptides

The polypeptides of the invention include the polypeptide of Table 1[SEQ ID NO:2] (in particular the mature polypeptide) as well aspolypeptides and fragments, particularly those which have the biologicalactivity of clpL, and also those which have at least 70% identity to thepolypeptide of Table 1 [SEQ ID NO:2] or the relevant portion, preferablyat least 80% identity to the polypeptide of Table 1 [SEQ ID NO:2], andmore preferably at least 90% similarity (more preferably at least 90%identity) to the polypeptide of Table 1 [SEQ ID NO:2] and still morepreferably at least 95% similarity (still more preferably at least 95%identity) to the polypeptide of Table 1 [SEQ ID NO:2] and also includeportions of such polypeptides with such portion of the polypeptidegenerally containing at least 30 amino acids and more preferably atleast 50 amino acids.

The invention also includes polypeptides of the formula set forth inTable 1 (D) wherein, at the amino terminus, X is hydrogen, and at thecarboxyl terminus, Y is hydrogen or a metal, R₁ and R₂ is any amino acidresidue, and n is an integer between 1 and 1000. Any stretch of aminoacid residues denoted by either R group, where R is greater than 1, maybe either a heteropolymer or a homopolymer, preferably a heteropolymer.

A fragment is a variant polypeptide having an amino acid sequence thatentirely is the same as part but not all of the amino acid sequence ofthe aforementioned polypeptides. As with clpL polypeptides fragments maybe “free-standing,” or comprised within a larger polypeptide of whichthey form a part or region, most preferably as a single continuousregion, a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of the amino acid sequence of Table 1 [SEQ ID NO:2], or ofvariants thereof, such as a continuous series of residues that includesthe amino terminus, or a continuous series of residues that includes thecarboxyl terminus. Degradation forms of the polypeptides of theinvention in a host cell, particularly a Staphylococcus aureus, are alsopreferred. Further preferred are fragments characterized by structuralor functional attributes such as fragments that comprise alpha-helix andalpha-helix forming regions, beta-sheet and beta-sheet-forming regions,turn and turn-forming regions, coil and coil-forming regions,hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, flexible regions, surface-forming regions,substrate binding region, and high antigenic index regions.

Also preferred are biologically active fragments which are thosefragments that mediate activities of clpL, including those with asimilar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Staphylococcusaureus or the ability to initiate, or maintain cause disease in anindividual, particularly a human.

Variants that are fragments of the polypeptides of the invention may beemployed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides of theinvention.

Polynucleotides

Another aspect of the invention relates to isolated polynucleotides,including the full length gene, that encode the clpL polypeptide havingthe deduced amino acid sequence of Table 1 [SEQ ID NO:2] andpolynucleotides closely related thereto and variants thereof.

Using the information provided herein, such as the polynucleotidesequence set out in Table 1 [SEQ ID NO:1], a polynucleotide of theinvention encoding clpL polypeptide may be obtained using standardcloning and screening methods, such as those for cloning sequencingchromosomal DNA fragments from bacteria using Staphylococcus aureus WCUH29 cells as starting material, followed by obtaining a full lengthclone. For example, to obtain a polynucleotide sequence of theinvention, such as the sequence given in Table 1 [SEQ ID NO:1],typically a library of clones of chromosomal DNA of Staphylococcusaureus WCUH 29 in E. coli or some other suitable host is probed with aradiolabeled oligonucleotide, preferably a 17-mer or longer, derivedfrom a partial sequence. Clones carrying DNA identical to that of theprobe can then be distinguished using stringent conditions. Bysequencing the individual clones thus identified with sequencing primersdesigned from the original sequence it is then possible to extend thesequence in both directions to determine the full gene sequence.Conveniently, such sequencing is performed using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989). (see in particular Screening ByHybridization 1.90 and Sequencing Denatured Double-Stranded DNATemplates 13.70). Illustrative of the invention, the polynucleotide setout in Table 1 [SEQ ID NO:1] was discovered in a DNA library derivedfrom Staphylococcus aureus WCUH 29.

The DNA sequence set out in Table 1 [SEQ ID NO:1] contains an openreading frame encoding a protein having about the number of amino acidresidues set forth in Table 1 [SEQ ID NO:2] with a deduced molecularweight that can be calculated using amino acid residue molecular weightvalues well known in the art. The polynucleotide of SEQ ID NO: 1,between nucleotide number 1 through number 2103 encodes the polypeptideof SEQ ID NO:2. The stop codon begins at nucleotide number 2104 of SEQID NO:1.

The clpL polypeptide of the invention is structurally related to otherproteins of the ATP dependent proteases family, as shown by the resultsof sequencing the DNA encoding clpL of the deposited strain. The proteinexhibits greatest homology to Lactococcus lactis ClpL protein amongknown proteins. The clpL polypeptide of Table 1 [SEQ ID NO:2] has about55% identity over its entire length and about 73% similarity over itsentire length with the amino acid sequence of Loctococcus lactis ClpLpolypeptide.

The invention provides a polynucleotide sequence identical over itsentire length to the coding sequence in Table 1 [SEQ ID NO:1]. Alsoprovided by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro- protein sequence. Thepolynucleotide may also contain non-coding sequences, including forexample, but not limited to non-coding 5′ and 3′ sequences, such as thetranscribed, non-translated sequences, termination signals, ribosomebinding sites, sequences that stabilize mRNA, introns, polyadenylationsignals, and additional coding sequence which encode additional aminoacids. For example, a marker sequence that facilitates purification ofthe fused polypeptide can be encoded. In certain embodiments of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc.Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA tag (Wilson et al.,Cell 37: 767 (1984). Polynucleotides of the invention also include, butare not limited to, polynucleotides comprising a structural gene and itsnaturally associated sequences that control gene expression.

A preferred embodiment of the invention is the polynucleotide ofcomprising nucleotide 1 to 2103 set forth in SEQ ID NO:1 of Table 1which encodes the clpL polypeptide.

The invention also includes polynucleotides of the formula set forth inTable 1 (C) wherein, at the 5′ end of the molecule, X is hydrogen, andat the 3′ end of the molecule, Y is hydrogen or a metal, R₁ and R₂ isany nucleic acid residue, and n is an integer between 1 and 1000. Anystretch of nucleic acid residues denoted by either R group, where isgreater than 1, may be either a heteropolymer or a homopolymer,preferably a heteropolymer.

The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the staphylococcus aureus clpL havingthe amino acid sequence set out in Table 1 [SEQ ID NO:2]. The term alsoencompasses polynucleotides that include a single continuous region ordiscontinuous regions encoding the polypeptide (for example, interruptedby integrated phage or an insertion sequence or editing) together withadditional regions, that also may contain coding and/or non-codingsequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptide having thededuced amino acid sequence of Table 1 [SEQ ID NO:2]. Variants that arefragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingclpL variants, that have the amino acid sequence of clpL polypeptide ofTable 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1 to 3, 2, 1 orno amino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, that do not alter the properties and activitiesof clpL.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over the entire length to a polynucleotideencoding clpL polypeptide having the amino acid sequence set out inTable 1 [SEQ ID NO:2], a polynucleotides that are complementary to suchpolynucleotides. Alternatively most highly preferred are polynucleotidesthat comprise a region that is at least 80% identical over its entirelength to a polynucleotide encoding clpL polypeptide of the depositedstrain and polynucleotides complementary thereto. In this regard,polynucleotides at least 90% identical over their entire length to thesame are particularly preferred, and among these particularly preferredpolynucleotides, those with at least 95% are especially preferred.Furthermore, those with at least 97% are highly preferred among thosewith at least 95%, and among these those with at least 98% and at least99% are particularly highly preferred, with at least 99% being the morepreferred.

Preferred embodiments are polynucleotides that encode polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by the DNA of Table 1 [SEQ ID NO:1].

The invention further relates to polynucleotides that hybridize to theherein above-described sequences. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms “stringent conditions” and “stringent hybridizationconditions ” mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the hybridization support in 0.1×SSC at about 65° C. Hybridization and wash conditions are well known andexemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter11 therein.

The invention also provides a polynucleotide consisting essentially of apolynucleotide sequence obtainable by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set fourth inSEQ ID NO:1 under stringent hybridization conditions with a probe havingthe sequence of said polynucleotide sequence set forth in SEQ ID NO:1 ora fragment thereof; and isolating said DNA sequence. Fragments usefulfor obtaining such a polynucleotide include, for example, probes andprimers described else where herein.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe of RNA, cDNA and genomic DNAto isolate full-length cDNAs and genomic clones encoding clpL and toisolate cDNA and genomic clones of other genes that have a high sequencesimilarity to the clpL gene. Such probes generally will comprise atleast 15 bases. Preferably, such probes will have at least 30 bases andmay have at least 50 bases. Particularly preferred probes will have atleast 30 bases and will have 50 bases or less.

For example, the coding region of the clpL gene may be isolated byscreening using the DNA sequence provided in SEQ ID NO: 1 to synthesizean oligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the invention is then used to screena library of cDNA, genomic DNA or mRNA to determine which members of thelibrary the probe hybridizes to.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for disease, particularly human disease,as further discussed herein relating to polynucleotide assays.

Polynucleotides of the invention that are oligonucleotides derived fromthe sequences of SEQ ID NOS:1 and/or 2 may be used in the processesherein as described, but preferably for PCR, to determine whether or notthe polynucleotides identified herein in whole or in part aretranscribed in bacteria in infected tissue. It is recognized that suchsequences will also have utility in diagnosis of the stage of infectionand type of infection the pathogen has attained.

The invention also provides polynucleotides that may encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, host cells, expression

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof or polynucleotides ofthe invention. Introduction of a polynucleotide into the host cell canbe effected by methods described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), suchas, calcium phosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

Representative examples of appropriate hosts include bacterial cells,such as steptrococi, staphylococci, enterococci E. coli, streptomycesand Bacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic Assays

This invention is also related to the use of the clpL polynucleotides ofthe invention for use as diagnostic reagents. Detection of clpL in aeukaryote, particularly a mammal, and especially a human, will provide adiagnostic method for diagnosis of a disease. Eukaryotes (herein also“individual(s)”), particularly mammals, and especially humans,particularly those infected or suspected to be infected with an organismcomprising the clpL gene may be detected at the nucleic acid level by avariety of techniques.

Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA or cDNA may also be used in the same ways. Usingamplification, characterization of the species and strain of prokaryotepresent in an individual, may be made by an analysis of the genotype ofthe prokaryote gene. Deletions and insertions can be detected by achange in size of the amplified product in comparison to the genotype ofa reference sequence. Point mutations can be identified by hybridizingamplified DNA to labeled clpL polynucleotide sequences. Perfectlymatched sequences can be distinguished from mismatched duplexes by RNasedigestion or by differences in melting temperatures. DNA sequencedifferences may also be detected by alterations in the electrophoreticmobility of the DNA fragments in gels, with or without denaturingagents, or by direct DNA sequencing, See, e.g., Myers et al., Science,230: 1242 (1985). Sequence changes at specific locations also may berevealed by nuclease protection assays, such as RNase and S1 protectionor a chemical cleavage method. See, e.g., Cotton et al., Proc. Natl.Acad. Sci., U.S.A., 85:4397-4401 (1985).

Cells carrying mutations or polymorphisms in the gene of the inventionmay also be detected at the DNA level by a variety of techniques, toallow for serotyping, for example. For example, RT-PCR can be used todetect mutations. It is particularly preferred to used RT-PCR inconjunction with automated detection systems, such as, for example,GeneScan. RNA or cDNA may also be used for the same purpose, PCR orRT-PCR. As an example, PCR primers complementary to a nucleic acidencoding clpL can be used to identify and analyze mutations. Examples ofrepresentative primers are shown below in Table 2.

TABLE 2 Primers for amplification of clpL polynucleotides SEQ ID NOPRIMER SEQUENCE 3 5′-ATGAATAACG GTTTTTTCAA TAGC-3′ 4 5′-TTATTTACCTTTTACGACTA ATTC-3′

The invention further provides these primers with 1, 2, 3 or 4nucleotides removed from the 5′ and/or the 3′ end. These primers may beused for, among other things, amplifying clpL DNA isolated from a samplederived from an individual. The primers may be used to amplify the geneisolated from an infected individual such that the gene may then besubject to various techniques for elucidation of the DNA sequence. Inthis way, mutations in the DNA sequence may be detected and used todiagnose infection and to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStaphylococcus aureus, and most preferably disease, such as, infectionsof the upper respiratory tract (e.g., otitis media, bacterialtracheitis, acute epiglottitis, thyroiditis), low respiratory (e.g.,empyema, lung abscess), cardiac (e.g., infective endocarditis),gastrointestinal (e.g., secretory diarrhoea, splenic abscess,retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g.,blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal andorbital cellulitis, darcryocystitis), kidney and urinary tract (e.g.,epididymitis, intrarenal and perinephric abscess, toxic shock syndrome),skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis,wound infection, bacterial myositis) bone and joint (e.g., septicarthritis, osteomyelitis), comprising determining from a sample derivedfrom an individual a increased level of expression of polynucleotidehaving the sequence of Table 1 [SEQ ID NO: 1]. Increased or decreasedexpression of clpL polynucleotide can be measured using any on of themethods well known in the art for the quantation of polynucleotides,such as, for example, amplification, PCR, RT-PCR, RNase protection,Northern blotting and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of clpL protein compared to normal controltissue samples may be used to detect the presence of an infection, forexample. Assay techniques that can be used to determine levels of a clpLprotein, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

Antibodies

The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. “Antibodies” as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

Antibodies generated against the polypeptides of the invention can beobtained by administering the polypeptides or epitope-bearing fragments,analogues or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique knownin the art that provides antibodies produced by continuous cell linecultures can be used. Examples include various techniques, such as thosein Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor etal., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-clpL or from naive libraries (McCafferty,J. et al., (1990), Nature 348: 552-554; Marks, J. et al., (1992)Biotechnology 10, 779-783). The affinity of these antibodies can also beimproved by chain shuffling (Clackson, T. et al., (1991) Nature 352,624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope—terms ‘bispecific’ antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides to purify the polypeptides byaffinity chromatography.

Thus, among others, antibodies against clpL-polypeptide may be employedto treat infections, particularly bacterial infections and especiallydisease, such as, infections of the upper respiratory tract (e.g.,otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis),lower respiratory (e.g., empyema, lung abscess), cardiac (e.g.,infective endocarditis), gastrointestinal (e.g., secretory diarrhoea,splenic abscess, retroperitoneal abscess), CNS (e.g., cerebral abscess),eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis,preseptal and orbital cellulitis, darcryocystitis), kidney and urinarytract (e.g., epididymitis, intrarenal and perinephric abscess, toxicshock syndrome), skin (e.g., impetigo, folliculitis, cutaneousabscesses, cellulitis, wound infection, bacterial myositis) bone andjoint (e.g., septic arthritis, osteomyelitis).

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect of theinvention. The term “antigenically equivalent derivative” as used hereinencompasses a polypeptide or its equivalent which will be specificallyrecognized by certain antibodies which, when raised to the protein orpolypeptide according to the invention, interfere with the immediatephysical interaction between pathogen and mammalian host. The term“immunologically equivalent derivative” as used herein encompasses apeptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rate or chicken. The fusion proteinmay provide stability to the polypeptide. The antigen may be associated,for example by conjugation, with an immunogenic carrier protein forexample bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be “humanized”; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal., (1991) Biotechnology 9, 266-273.

The use of a polypeptide of the invention in genetic immunization willpreferably employ a suitable delivery method such as direct injection ofplasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992, 1:363,Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem. 1989:264, 16985), coprecipitation of DNA with calcium phosphate (Benvenisty &Reshef, PNAS U.S.A., 1986:83,9551), encapsulation of DNA in variousforms of liposomes (Kaneda et al., Science 1989:243,375), particlebombardment (Tang et al., Nature 1992, 356:152, Eisenbraun et al., DNACell Biol 1993, 12:791) and in vivo infection using cloned retroviralvectors (Seeger et al., PNAS U.S.A. 1984:81,5849).

Antagonists and agonists—assays and molecules

Polypeptides of the invention also may be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See, e.g., Coligan et al., CurrentProtocols in Immunology 1(2):Chapter 5 (1991).

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action of clpLpolypeptides or polynucleotides, particularly those compounds that arebacteriostatic and/or bacteriocidal. The method of screening may involvehigh-throughput techniques. For example, to screen for agonists orantagonists, a synthetic reaction mix, a cellular compartment, such as amembrane, cell envelope or cell wall, or a preparation of any thereof,comprising clpL polypeptide and a labeled substrate or ligand of suchpolypeptide is incubated in the absence or the presence of a candidatemolecule that may be a clpL agonist or antagonist. The ability of thecandidate molecule to agonize or antagonize the clpL polypeptide isreflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of clpL polypeptide aremost likely to be good antagonists. Molecules that bind well andincrease the rate of product production from substrates are agonists.Detection of the rate or level of production of product from substratemay be enhanced by using a reporter system. Reporter systems that may beuseful in this regard include but are not limited to colorimetriclabeled substrate converted into product, a reporter gene that isresponsive to changes in clpL polynucleotide or polypeptide activity,and binding assays known in the art.

Another example of an assay for clpL antagonists is a competitive assaythat combines clpL and a potential antagonist with clpL-bindingmolecules, recombinant clpL binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. The clpL molecule can be labeled,such as by radioactivity or a colorimetric compounds, such that thenumber of clpL molecules bound to a binding molecule or converted toproduct can be determined accurately to assess the effectiveness of thepotential antagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, a peptide, apolypeptide such as closely related protein or antibody that binds thesame sites on a binding molecule, such as a binding molecule, withoutinducing clpL-induced activities, thereby preventing the action of clpLby excluding clpL from binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of clpL.

Each of the DNA sequences provided herein may be used in the discoveryand development of antibacterial compounds. The encoded protein, uponexpression, can be used as a target for the screening of antibacterialdrugs. Additionally, the DNA sequences encoding the amino terminalregions of the encoded protein or Shine-Delgarno or other translationfacilitating sequences of the respective mRNA can be used to constructantisense sequences to control the expression of the coding sequence ofinterest.

The invention also provides the use of the polypeptide, polynucleotideor inhibitor of the invention to interfere with the initial physicalinteraction between a pathogen and mammalian host responsible forsequelae of infection. In particular the molecules of the invention maybe used: in the prevention of adhesion of bacteria, in particular grampositive bacteria, to mammalian extracellular matrix proteins onin-dwelling devices or to extracellular matrix proteins in wounds; toblock clpL protein-mediated mammalian cell invasion by, for example,initiating phosphorylation of mammalian tyrosine kinases (Rosenshine etal., Infect. Immun. 60:2211 (1992); to block bacterial adhesion betweenmammalian extracellular matrix proteins and bacterial clpL proteins thatmediate tissue damage and; to block the normal progression ofpathogenesis in infections initiated other than by the implantation ofin-dwelling devices or by other surgical techniques.

The antagonists and agonists of the invention may be employed, forinstance, to inhibit and treat disease, such as, infections of the upperrespiratory tract (e.g., otitis media, bacterial tracheitis, acuteepiglottis, thryoiditis), lower respiratory (e.g., empyema, lungabscess), cardiac (e.g., infective endocarditis), gastrointestinal(e.g., secretory diarrhoea, splenic abscess, retroperitoneal abscess),CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal and orbital cellulitis,darcryocystitis), kidney and urinary tract (e.g., epididymitis,intrarenal and perinephric abscess, toxic shock syndrome), skin (e.g.,impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis) bone and joint (e.g., septic arthritis,osteomyelitis).

Helicobacter pylori (herein H. pylori) bacteria infect the stomaches ofover one-third of the world's population causing stomach cancer, ulcers,and gastritis (International Agency for Research on Cancer (1994)Schistosomes, Liver Flukes and Heliobacter Pylori (International Agencyfor Research on Cancer, Lyon, France;http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the international Agencyfor Research on Cancer recently recognized a cause-and-effectrelationship between H. pylori and gastric adenocarcinoma, classifyingthe bacterium as a Group I (definite) carcinogen. Preferredantimicrobial compounds of the invention (agonists and antagonists ofclpL) found using screens provided by the invention, particularlybroad-spectrum antibiotics, should be useful in the treatment of H.pylori infection. Such treatment should decrease the advent of H.pylori-induced cancers, such as gastrointestinal carcinoma. Suchtreatment should also cure gastric ulcers and gastritis.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with clpL, or a fragment or variantthereof, adequate to produce antibody and/or T cell immune response toprotect said individual from infection, particularly bacterial infectionand most particularly Staphylococcus aureus infection. Also provided aremethods whereby such immunological response slows bacterial replication.Yet another aspect of the invention relates to a method of inducingimmunological response in an individual which comprises delivering tosuch individual a nucleic acid vector to direct expression of clpL, or afragment or a variant thereof, for expressing clpL, or a fragment or avariant thereof in vivo in order to induce an immunological response,such as, to produce antibody and/or T cell immune response, including,for example, cytokine-producing T cells or cytotoxic T cells, to protectsaid individual from disease, whether that disease is alreadyestablished within the individual or not. One way of administering thegene is by accelerating it into the desired cells as a coating onparticles or otherwise.

Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid,or a DNA/RNA hybrid.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into an individual capable or havinginduced within it an immunological response, induces an immunologicalresponse in such individual to a clpL or protein coded therefrom,wherein the composition comprises a recombinant clpL or protein codedtherefrom comprising DNA which codes for and expresses an antigen ofsaid clpL or protein coded therefrom. The immunological response may beused therapeutically or prophylactically and may take the form ofantibody immunity or cellular immunity such as that arising from CTL orCD4+ T cells.

A clpL polypeptide or a fragment thereof may be fused with co-proteinwhich may not by itself produce antibodies, but is capable ofstabilizing the first protein and producing a fused protein which willhave immunogenic and protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such aslipoprotein D from Hemophilus influenzae, Glutathione-S-transferase(GST) or beta-galactosidase, relatively large co-proteins whichsolubilize the protein and facilitate production and purificationthereof. Moreover, the co-protein may act as an adjuvant in the sense ofproviding a generalized stimulation of the immune system. The co-proteinmay be attached to either the amino or carboxy terminus of the firstprotein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides of polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Staphylococcus aureus will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStaphylococcus aureus infection, in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused, e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation insotonic with the bodily fluid, preferably the blood, ofthe individual; and aqueous and non-aqueous sterile suspensions whichmay include suspending agents or thickening agents. The formulations maybe presented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certain clpLprotein, it is to be understood that this covers fragments of thenaturally occurring protein and similar proteins with additions,deletions or substitutions which do not substantially affect theimmunogenic properties of the recombinant protein.

Compositions, kits and administration

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or their agonists or antagonists.The polypeptides of the invention may be employed in combination with anon-sterile or sterile carrier or carriers for use with cells, tissuesor organisms, such as a pharmaceutical carrier suitable foradministration to a subject. Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of a polypeptide ofthe invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the invention may be employed aloneor in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers manyconstituted from about 1% to about 98% by weight of the formulation;more usually they will constitute up to about 80% by weight of theformulation.

For administration of mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of theinvention.

In-dwelling devices includes surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStaphyloccocus aureus wound infections.

Many orthopaedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significnat morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit does for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Each reference disclosed herein is incorporated by reference herein inits entirety. Any patent application to which this application claimspriority is also incorporated by reference herein it its entirety.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1 Strain selection, Library Production and Sequencing

The polynucleotide having the DNA sequence given in SEQ ID NO:1 wasobtained from a library of clones of chromosomal DNA of Staphylococcusaureus in E. coli. The sequencing data from two or more clonescontaining overlapping Staphylococcus aureus DNAs was used to constructthe contiguous DNA sequence in SEQ ID NO:1. Libraries may be prepared byroutine methods, for example:

Methods 1 and 2 below.

Total cellular DNA is isolated from Staphylococcus aureus WCUH 29according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E. coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E. coli infected with the packagedlibrary. The library is amplified by standard procedures.

Example 2 The determination of expression during infection of a genefrom Staphylococcus aureus

Necrotic fatty tissue from a four day groin infection of Staphylococcusaureus WCUH29 in the mouse is efficiently disrupted and processed in thepresence of chaotropic agents and RNAase inhibitor to provide a mixtureof animal and bacterial RNA. The optimal conditions for disruption andprocessing to give stable preparations and high yields of bacterial RNAare followed by the use of hybridisation to a radiolabelledoligonucleotide specific to Staphylococcus aureus 16S RNA on Northernblots. The RNAase free, DNAase free, DNA and protein free preparationsof RNA obtained are suitable for Reverse Transcription PCR (RT-PCR)using unique primer pairs designed from the sequence of each gene ofStaphylococcus aureus WCUH29.

a) Isolation of tissue infected with Staphylococcus aureus WCUH29 from amouse animal model of infection

10 ml. volumes of sterile nutrient broth (No. 2Oxoid) are seeded withisolated, individual colonies of Staphylococcus aureus WCUH29 from anagar culture plate. The cultures are incubated aerobically (staticculture) at 37° C. for 16-20 hours. 4 week old mice (female, 18 g-22 g,strain MF1) are each infected by subcutaneous injection of 0.5 ml. ofthis broth culture of Staphylococcus aureus WCUH29 (diluted in broth toapproximately 108 cfu/ml.) into the anterior, right lower quadrant(groin area). Mice should be monitored regularly during the first 24hours are infection, then daily until termination of study. Animals withsigns of systemic infection, i.e. lethargy, ruffled appearance,isolation from group, should be monitored closely and if signs progressto moribundancy, the animal should be culled immediately.

Visible external signs of lesion development will be seen 24-48 h afterinfection. Examination of the abdomen of the animal will show the raisedoutline of the abscess beneath the skin. The localized lesion shouldremain in the right lower quadrant, but may occasionally spread to theleft lower quadrant, and superiorly to the thorax. On occasions, theabscess may rupture through the overlying skin layers. In such cases theaffected animal should be culled immediately and the tissues sampled ifpossible. Failure to cull the animal may result in the necrotic skintissue overlying the abscess being sloughed off, exposing the abdominalmuscle wall.

Approximately 96 hours after infection, animals are killed using carbondioxide asphyxiation. To minimize delay between death and tissueprocessing/storage, mice should be killed individually rather than ingroups. The dead animal is placed onto its back and the fur swabbedliberally with 70% alcohol. An initial incision using scissors is madethrough the skin of the abdominal left lower quadrant, travellingsuperiorly up to, then across the thorax. The incision is completed bycutting inferiorly to the abdominal lower right quadrant. Care should betaken not to penetrate the abdominal wall. Holding the skin flap withforceps, the skin is gently pulled way from the abdomen. The exposedabscess, which covers the peritoneal wall but generally does notpenetrate the muscle sheet completely, is excised, taking care not topuncture the viscera.

The abscess/muscle sheet and other infected tissue may require cuttingin sections, prior to flash-freezing in liquid nitrogen, therebyallowing easier storage in plastic collecting vials.

b) Isolation of Staphylococcus aureus WCUH29 RNA from infection tissuesamples

4-6 infected tissue samples (each approx. 0.5-0.7 g) in 2 ml screw-captubes are removed from −80° C. storage into a dry ice ethanol bath. In amicrobiological safety cabinet the samples are disrupted individuallywhilst the remaining samples are kept cold in the dry ice ethanol bath.To disrupt the bacteria within the tissue sample 1 ml of TRIzol Reagent(Gibco BRL, Life Technologies) is added followed by enough 0.1 mmzirconia/silica beads to almost fill the tube, the lid is replacedtaking care not to get any beads into the screw thread so as to ensure agood seal and eliminate aerosol generation. The sample is thenhomogenised in a Mini-BeadBeater Type BX-4 (Biospec Products). Necroticfatty tissue is strain treated for 100 seconds at 5000 rpm in order toachieve bacterial lysis. In vivo grown bacteria require longer treatmentthan in vitro grown Staphylococcus aureus which are disrupted by a 30second bead-beat.

After bead-beating the tubes are chilled on ice before opening in afume-hood as heat generated during disruption may degrade the TRIzol andrelease cyanide.

200 microliters of chloroform is then added and the tubes shaken by handfor 15 seconds to ensure complete mixing. After 2-3 minutes at roomtemperature the tubes are spun down at 12,000×g, 4° C. for 15 minutesand RNA extraction is then continued according to the method given bythe manufacturers of TRIzol Reagent i.e.: The aqueous phase, approx 0.6ml, is transferred to a sterile eppendorf tube and 0.5 ml of isopropanolis added. After 10 minutes at room temperature the samples are spun at12,000×g, 4° C. for 10 minutes. The supernatant is removed and discardedthen the RNA pellet is washed with 1 ml 75% ethanol. A brief vortex isused to mix the sample before centrifuging at 7,500×g, 4° C. for 5minutes. The ethanol is removed and the RNA pellet dried under vacuumfor no more than 5 minutes. Samples are then resuspended by repeatedpipetting in 100 microliters of DEPC treated water, followed by 5-10minutes at 55° C. Finally, after at least 1 minute on ice, 200 units ofRnasin (Promega) is added.

RNA preparations are stored at −80° C. for up to one month. For longerterm storage the RNA precipitate can be stored at the wash stage of theprotocol in 75% ethanol for at least one year at −20° C.

Quality of the RNA isolated is assessed by running samples on 1% agarosegels. 1× TBE gels stained with ethidium bromide are used to visualizetotal RNA yields. To demonstrate the isolation of bacterial RNA from theinfected tissue 1×MOPS, 2.2 M formaldehyde gels are run and vacuumblotted to Hybond-N (Amersham). The blot is then hybridised with a 32 Plabelled oligonucleotide probe specific to 16 s rRNA of Staphylococcusaureus (K. Greisen, M. Loeffelholz, A. Purohit and D. Leong, J.Clin.(1994) Microbiol. 32 335-351). An oligonucleotide of the sequence:5′-GCTCCTAAAAGGTTACTCCACCGGC-3′ [SEQ ID NO:5] is used as a probe. Thesize of the hybridizing band is compared to that of control RNA isolatedfrom in vitro grown Staphylococcus aureus WCUH29 in the Northern blot.Correct sized bacterial 16s rRNA bands can be detected in total RNAsamples which show extensive degradation of the mammalian RNA whenvisualised on TBE gels.

c) The removal of DNA from Staphylococcus aureus WCUH29-derived RNA

DNA was removed from 73 microliter samples of RNA by a 15 minutetreatment on ice with 3 units of DNAaseI, amplification grade (GibcoBRL, Life Technologies) in the buffer supplied with the addition of 200units of Rnasin (Promega) in a final volume of 90 microliters.

The DNAase was inactivated and removed by treatment with TRIzol LSReagent (Gibco BRL, Life Technologies) according to the manufacturersprotocol. DNAase treated RNA was resuspended in 73 microliters of DEPCtreated water with the addition of Rnasin as described in Method 1.

d) The preparation of cDNA from RNA samples derived from infected tissue

10 microliter samples of DNAase treated RNA are reverse transcribedusing a SuperScript Preamplification System for First Strand cDNASynthesis kit (Gibco BRL, Life Technologies) according to themanufacturers instructions. 1 nanogram of random hexamers is used toprime each reaction. Controls without the addition of SuperScriptIIreverse transcriptase are also run. Both +/−RT samples are treated withRNaseH before proceeding to the PCR reaction.

e) The use of PCR to determine the presence of a bacterial cDNA species

PCR reactions are set up on ice in 0.2 ml tubes by adding the followingcomponents: 45 microliters PCR SUPERMIX (Gibco BRL, Life Technologies);1 microliter 50 mM MgCl2, to adjust final concentration to 2.5 mM; 1microliter PCR primers (optimally 18-25 basepairs in length and designedto possess similar annealing temperatures), each primer at 10 mM initialconcentration; and 2 microliters cDNA.

PCR reactions are run on a Perkin Elmer GeneAmp PCR System 9600 asfollows: 5 minutes at 95° C., then 50 cycles of 30 seconds each at 94°C., 42° C. and 72°0 C. followed by 3 minutes at 72° C. and then a holdtemperature of 4° C. (the number of cycles is optimally 30-50 todetermine the appearance or lack of a PCR product and optimally 8-30cycles if an estimation of the starting quantity of cDNA from the RTreaction is to be made); 10 microliter aliquots are then run out on 1%1× TBE gels stained with ethidium bromide with PCR product, if present,sizes estimated by comparison to a 100 bp DNA Ladder (Gibco BRL, LifeTechnologies). Alternatively if the PCR products are convenientlylabelled by the use of a labelled PCR primer (e.g. labelled at the 5′endwith a dye) a suitable aliquot of the PCR product is run out on apolyacrylamide sequencing gel and its presence and quantity detectedusing a suitable gel scanning system (e.g. ABI Prism™ 377 Sequencerusing GeneScan™ software as supplied by Perkin Elmer).

RT/PCR controls may include +/− reverse transcriptase reactions, 16srRNA primers or DNA specific primer pairs designed to produce PCRproducts from non-transcribed Staphylococcus aureus WCUH29 genomicsequences.

To test the efficiency of the primer pairs they are used in DNA PCR withStaphylococcus aureus WCUH29 total DNA. PCR reactions are set up and runas described above using approx. 1 microgram of DNA in place of the cDNAand 35 cycles of PCR.

Primer pairs which fail to give the predicted sized product in eitherDNA PCR or RT/PCR are PCR failures and as such are uninformative. Ofthose which give the correct size product with DNA PCR two classes aredistinguished in RT/PCR: 1.Genes which are not transcribed in vivoreproducibly fail to give a product in RT/PCR; and 2.Genes which aretranscribed in vivo reproducibly give the correct size product in RT/PCRand show a stronger signal in the +RT samples than the signal (if at allpresent) in −RT controls.

5 2106 base pairs nucleic acid single linear not provided 1 ATGAATAACGGTTTTTTCAA TAGCGACTTT GATTCAATTT TTCGAAGAAT GATGCAAGAT 60 ATGCAAGGTTCAAATCAAGT CGGTAACAAA AAGTACTATA TTAATGGTAA AGAAGTTTCA 120 CCTGAAGAACTAGCGCAACT CACACAACAA GGTAGCAATC AATCTGCTGA ACAAAGTGCG 180 CAAGCTTTTCAACAAGCAGC ACAAAGACAA CAAGGGCAAC AAGGTGGCAA CGGCAATTAT 240 TTAGAACAAATTGGTCGTAA CCTTACGCAA GAAGCACGTG ACGGTTTATT AGATCCAGTC 300 ATTGGTCGTGATAAAGAAAT TCAAGAAACT GCTGAAGTCT TAAGTAGACG AACTAAAAAC 360 AATCCTATATTAGTTGGAGA AGCTGGTGTT GGTAAAACTG CGATTGTTGA AGGTTTAGCA 420 CAGGCAATCGTTGAAGGAAA TGTACCAGCA GCAATCAAAG ACAAAGAAAT TATTTCTGTA 480 GATATTTCATCATTAGAAGC TGGAACGCAA TATCGTGGTG CTTTTGAAGA AAATATTCAA 540 AAATTAATCGAAGGTGTTAA ATCTTCACAA AATGCCGTAC TATTCTTTGA TGAAATCCAT 600 CAAATTATCGGTTCAGGTGC CACAGGAAGT GATTCAGGTA GCAAAGGGTT ATCTGATATT 660 TTGAAACCTGCATTAAGTCG TGGTGAGATT TCTATTATTG GTGCAACAAC ACAAGATGAA 720 TATCGAAACAATATTCTTAA AGATGCTGCA TTAACGCGCA GATTTAATGA AGTGCTTGTT 780 AATGAACCAAGCGCTAAAGA TACTGTTGAA ATTTTAAAAG GTATTCGCGA AAAATTCGAA 840 GAACACCATCAAGTAAAATT ACCAGATGAC GTATTAAAAG CATGTGTTGA CTTATCAATT 900 CAATATATTCCACAACGATT ATTACCAGAT AAAGCAATCG ATGTGTTAGA TATTACAGCA 960 GCACATTTATCTGCGCAAAG TCCAGCTGTC GATAAAGTTG AAACTGAAAA ACGAATTTCT 1020 GAATTAGAAAATGATAAACG TAAAGCAGTA AGTGCTGAAG AATATAAAAA AGCTGACGAC 1080 ATTCAAAATGAAATCAAATC ATTACAAGAT AAATTAGAAA ACAGTAATGG TGAACATACT 1140 GCTGTTGCTACAGTTCATGA TATTTCAGAT ACTATTCAAC GATTAACTGG CATTCCAGTT 1200 TCTCAAATGGATGATAACGA TATTGAACGT TTAAAAAATA TTTCTAATCG TTTAAGAAGT 1260 AAAATCATAGGTCAAGATCA AGCTGTAGAA ATGGTTTCAC GTGCAATTCG CCGTAATCGC 1320 GCTGGGTTTGATGACGGCAA CCGTCCAATT GGTAGTTTCT TATTTGTCGG CCCTACTGGT 1380 GTTGGTAAAACAGAGCTTGC TAAACAATTA GCAATTGATC TATTTGGTAA TAAAGATGCA 1440 CTTATTCGACTTGATATGAG TGAATATAGT GACACAACAG CTGTTTCAAA AATGATTGGT 1500 ACAACTGCTGGTTATGTCGG TTATGATGAC AATTCAAATA CGTTAACTGA AAAAGTACGC 1560 CGTAATCCATACTCAGTCAT TCTATTTGAT GAAATCGAAA AAGCAAATCC ACAAATTTTA 1620 ACATTGTTATTACAAGTAAT GGATGATGGT AATTTGACTG ATGGTCAAGG TAATGTCATC 1680 AACTTTAAAAATACAATTAT TATTTGTACA TCAAATGCTG GCTTTGGCAA TGGCAATGAC 1740 GCTGAAGAAAAAGATATTAT GCACGAAATG AAAAAATTCT TCCGCCCTGA ATTCCTTAAC 1800 CGCTTCAACGGCATCGTTGA ATTCTTACAT TTAGATAAAG ATGCATTGCA AGATATCGTC 1860 AACTTATTATTAGACGATGT ACAAGTTACA TTAGATAAAA AAGGTATTAC GATGGACGTT 1920 TCTCAAGATGCGAAAGATTG GTTAATTGAA GAAGGCTATG ATGAAGAATT AGGTGCACGT 1980 CCATTAAGACGTATTGTTGA ACAGCAAGTA CGTGACAAAA TTACAGATTA TTATCTAGAT 2040 CATACAGACGTTAAACATGT GGATATAGAT GTTGAGGATA ACGAATTAGT CGTAAAAGGT 2100 AAATAA 2106701 amino acids amino acid double linear not provided 2 Met Asn Asn GlyPhe Phe Asn Ser Asp Phe Asp Ser Ile Phe Arg Arg 1 5 10 15 Met Met GlnAsp Met Gln Gly Ser Asn Gln Val Gly Asn Lys Lys Tyr 20 25 30 Tyr Ile AsnGly Lys Glu Val Ser Pro Glu Glu Leu Ala Gln Leu Thr 35 40 45 Gln Gln GlySer Asn Gln Ser Ala Glu Gln Ser Ala Gln Ala Phe Gln 50 55 60 Gln Ala AlaGln Arg Gln Gln Gly Gln Gln Gly Gly Asn Gly Asn Tyr 65 70 75 80 Leu GluGln Ile Gly Arg Asn Leu Thr Gln Glu Ala Arg Asp Gly Leu 85 90 95 Leu AspPro Val Ile Gly Arg Asp Lys Glu Ile Gln Glu Thr Ala Glu 100 105 110 ValLeu Ser Arg Arg Thr Lys Asn Asn Pro Ile Leu Val Gly Glu Ala 115 120 125Gly Val Gly Lys Thr Ala Ile Val Glu Gly Leu Ala Gln Ala Ile Val 130 135140 Glu Gly Asn Val Pro Ala Ala Ile Lys Asp Lys Glu Ile Ile Ser Val 145150 155 160 Asp Ile Ser Ser Leu Glu Ala Gly Thr Gln Tyr Arg Gly Ala PheGlu 165 170 175 Glu Asn Ile Gln Lys Leu Ile Glu Gly Val Lys Ser Ser GlnAsn Ala 180 185 190 Val Leu Phe Phe Asp Glu Ile His Gln Ile Ile Gly SerGly Ala Thr 195 200 205 Gly Ser Asp Ser Gly Ser Lys Gly Leu Ser Asp IleLeu Lys Pro Ala 210 215 220 Leu Ser Arg Gly Glu Ile Ser Ile Ile Gly AlaThr Thr Gln Asp Glu 225 230 235 240 Tyr Arg Asn Asn Ile Leu Lys Asp AlaAla Leu Thr Arg Arg Phe Asn 245 250 255 Glu Val Leu Val Asn Glu Pro SerAla Lys Asp Thr Val Glu Ile Leu 260 265 270 Lys Gly Ile Arg Glu Lys PheGlu Glu His His Gln Val Lys Leu Pro 275 280 285 Asp Asp Val Leu Lys AlaCys Val Asp Leu Ser Ile Gln Tyr Ile Pro 290 295 300 Gln Arg Leu Leu ProAsp Lys Ala Ile Asp Val Leu Asp Ile Thr Ala 305 310 315 320 Ala His LeuSer Ala Gln Ser Pro Ala Val Asp Lys Val Glu Thr Glu 325 330 335 Lys ArgIle Ser Glu Leu Glu Asn Asp Lys Arg Lys Ala Val Ser Ala 340 345 350 GluGlu Tyr Lys Lys Ala Asp Asp Ile Gln Asn Glu Ile Lys Ser Leu 355 360 365Gln Asp Lys Leu Glu Asn Ser Asn Gly Glu His Thr Ala Val Ala Thr 370 375380 Val His Asp Ile Ser Asp Thr Ile Gln Arg Leu Thr Gly Ile Pro Val 385390 395 400 Ser Gln Met Asp Asp Asn Asp Ile Glu Arg Leu Lys Asn Ile SerAsn 405 410 415 Arg Leu Arg Ser Lys Ile Ile Gly Gln Asp Gln Ala Val GluMet Val 420 425 430 Ser Arg Ala Ile Arg Arg Asn Arg Ala Gly Phe Asp AspGly Asn Arg 435 440 445 Pro Ile Gly Ser Phe Leu Phe Val Gly Pro Thr GlyVal Gly Lys Thr 450 455 460 Glu Leu Ala Lys Gln Leu Ala Ile Asp Leu PheGly Asn Lys Asp Ala 465 470 475 480 Leu Ile Arg Leu Asp Met Ser Glu TyrSer Asp Thr Thr Ala Val Ser 485 490 495 Lys Met Ile Gly Thr Thr Ala GlyTyr Val Gly Tyr Asp Asp Asn Ser 500 505 510 Asn Thr Leu Thr Glu Lys ValArg Arg Asn Pro Tyr Ser Val Ile Leu 515 520 525 Phe Asp Glu Ile Glu LysAla Asn Pro Gln Ile Leu Thr Leu Leu Leu 530 535 540 Gln Val Met Asp AspGly Asn Leu Thr Asp Gly Gln Gly Asn Val Ile 545 550 555 560 Asn Phe LysAsn Thr Ile Ile Ile Cys Thr Ser Asn Ala Gly Phe Gly 565 570 575 Asn GlyAsn Asp Ala Glu Glu Lys Asp Ile Met His Glu Met Lys Lys 580 585 590 PhePhe Arg Pro Glu Phe Leu Asn Arg Phe Asn Gly Ile Val Glu Phe 595 600 605Leu His Leu Asp Lys Asp Ala Leu Gln Asp Ile Val Asn Leu Leu Leu 610 615620 Asp Asp Val Gln Val Thr Leu Asp Lys Lys Gly Ile Thr Met Asp Val 625630 635 640 Ser Gln Asp Ala Lys Asp Trp Leu Ile Glu Glu Gly Tyr Asp GluGlu 645 650 655 Leu Gly Ala Arg Pro Leu Arg Arg Ile Val Glu Gln Gln ValArg Asp 660 665 670 Lys Ile Thr Asp Tyr Tyr Leu Asp His Thr Asp Val LysHis Val Asp 675 680 685 Ile Asp Val Glu Asp Asn Glu Leu Val Val Lys GlyLys 690 695 700 24 base pairs nucleic acid single linear not provided 3ATGAATAACG GTTTTTTCAA TAGC 24 24 base pairs nucleic acid single linearnot provided 4 TTATTTACCT TTTACGACTA ATTC 24 25 base pairs nucleic acidsingle linear not provided 5 GCTCCTAAAA GGTTACTCCA CCGGC 25

What is claimed is:
 1. An isolated polynucleotide segment comprising anucleic acid sequence that encodes a polypeptide comprising the aminoacid sequence of SEQ ID NO:2, wherein the nucleic acid sequence is notgenomic DNA.
 2. A vector comprising the isolated polynucleotide segmentof claim
 1. 3. An isolated host cell comprising the vector of claim 2.4. A process for producing a polypeptide comprising culturing the hostcell of claim 3 under conditions sufficient for the production of thepolypeptide, wherein the polypeptide comprises SEQ ID NO:2.
 5. Anisolated polynucleotide segment comprising SEQ ID NO:1.
 6. A vectorcomprising the isolated polynucleotide segment of claim
 5. 7. Anisolated host cell comprising the vector of claim
 6. 8. A process forproducing a polypeptide comprising culturing the host cell of claim 7under conditions sufficient for the production of the polypeptide,wherein the polypeptide comprises SEQ ID NO:2.
 9. An isolatedpolynucleotide comprising a nucleic acid sequence which encodes apolypeptide consisting of the amino acid sequence of SEQ ID NO:2,wherein the nucleic acid sequence is not genomic DNA.
 10. A vectorcomprising the isolated polynucleotide of claim
 9. 11. An isolated hostcell comprising the vector of claim
 10. 12. A process for producing apolypeptide comprising culturing the host cell of claim 11 underconditions sufficient for the production of the polypeptide, wherein thepolypeptide consists of SEQ ID NO:2.
 13. An isolated polynucleotidesegment comprising the full complement of the nucleotide sequence ofclaim 1, wherein the full complement is not genomic DNA and wherein thefull complement detects Staphylococcus aureus by hybridization.
 14. Anisolated polynucleotide segment comprising the full complement of thenucleotide sequence of claim 9, wherein the full complement is notgenomic DNA and wherein the full complement detects Staphylococcusaureus by hybridization.